Detection of IL35+ Exosomes as a Marker for Peripheral Tolerance

检测 IL35 外泌体作为外周耐受性的标志物

基本信息

批准号：
9978316
负责人：
Jeremy A Sullivan
金额：
$ 7.75万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9978316
关键词：
Address Animals Antibodies Antigens Ascites Autoantigens Autoimmune Process Biological Assay Blood CD81 gene Cell Culture Techniques Cells Control Animal Culture Media Data Delayed Hypersensitivity Detection Effector Cell Engineering Enzyme-Linked Immunosorbent Assay Failure Family Future Hour Human Human Herpesvirus 4 Immune Immune response Immunize Immunology Immunosuppression Inflammatory Response Interleukin-12 Interleukins Knockout Mice Label Lead Liquid substance LoxP-flanked allele Lymph Lymphocyte Malignant Neoplasms Measures Mediating Membrane Mus Physiological Production Property Protein Subunits Proteins Protocols documentation Regulation Regulatory T-Lymphocyte Reporter Reporting SCID Mice Serum Swelling TNFSF5 gene Testing Tetanus Toxoid Therapeutic Transfusion Transmission Electron Microscopy Transplant Recipients Transplantation cell transformation cytokine design exosome extracellular vesicles member mouse model neutralizing antibody nonhuman primate peripheral tolerance response transplant model

Address Animals Antibodies Antigens Ascites Autoantigens Autoimmune Process Biological Assay Blood CD81 gene Cell Culture Techniques Cells Control Animal Culture Media Data Delayed Hypersensitivity Detection Effector Cell Engineering Enzyme-Linked Immunosorbent Assay Failure Family Future Hour Human Human Herpesvirus 4 Immune Immune response Immunize Immunology Immunosuppression Inflammatory Response Interleukin-12 Interleukins Knockout Mice Label Lead Liquid substance LoxP-flanked allele Lymph Lymphocyte Malignant Neoplasms Measures Mediating Membrane Mus Physiological Production Property Protein Subunits Proteins Protocols documentation Regulation Regulatory T-Lymphocyte Reporter Reporting SCID Mice Serum Swelling TNFSF5 gene Testing Tetanus Toxoid Therapeutic Transfusion Transmission Electron Microscopy Transplant Recipients Transplantation cell transformation cytokine design exosome extracellular vesicles member mouse model neutralizing antibody nonhuman primate peripheral tolerance response transplant model

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项目摘要

ABSTRACT Interleukin 35 (IL35) has emerged as a potent immuno-suppressive cytokine in cancer, auto-immune and transplant immunology. A member of the IL12 family of cytokines, IL35 is a heterodimeric cytokine composed of the protein subunits Epstein Barr Virus Induced 3 (Ebi3) and p35 and is produced and secreted predominantly by regulatory T cells (Tregs). While IL35 has been implicated in the regulation of a number of cellular immune responses, there still exists significant debate as to whether the cytokine actually exists. This debate is predicated on the fact that attempts to isolate IL35 from blood, physiological solutions like ascites or lymph, or even cell culture supernatants have failed. We have recently immune-precipitated both Ebi3 and p35 with an antibody to the tetraspanin CD81 from mouse lymphocytes. Tetraspanins are membrane spanning proteins associated with the formation of the small, extracellular vesicles, exosomes. This interesting observation prompted us to examine whether IL35 exists as Ebi3 and p35 proteins associated with tetraspanins and secreted and acquired as an exosome. To test the idea that IL35 exists as an exosome dependent cytokine, we designed the following specific aims: Specific Aim 1: To test whether exosomes isolated from serum of tolerized groups of tetraspanin knockout mice (CD81, CD63, CD9) lose Ebi3 and p35 positive exosomes. Specific Aim 2: To examine whether lymphocytes from tolerized-CD81, CD63 or CD9 knockout mice produce and release functional IL35+ immuno-suppressive exosomes. The successful completion of the outlined specific aims in this proposal will fill a significant gap in our understanding of the cytokine IL35, and will ideally lead to future applications that further our understanding of the therapeutic implications of IL35 for humans.

抽象的白介素35（IL35）已成为癌症，自动免疫和移植免疫学。 IL12细胞因子家族的成员IL35是组成的异二聚体细胞因子蛋白质亚基的爱泼斯坦巴尔病毒诱导3（EBI3）和p35，并生产和分泌主要由调节性T细胞（Tregs）。虽然IL35与许多人有关细胞免疫反应，关于细胞因子是否真正存在的问题仍然存在重大争论。这辩论是基于以下事实：试图将IL35与血液，生理解决方案（如腹水）或淋巴，甚至细胞培养上清液都失败了。我们最近对EBI3和p35进行了免疫沉淀来自小鼠淋巴细胞的四跨果蛋白CD81的抗体。四跨果蛋白是膜的与小细胞外囊泡形成相关的蛋白质。这个有趣的观察促使我们检查IL35是否以EBI3和p35蛋白相关四翼烷蛋白并被分泌并作为外泌体获得。测试IL35作为外泌体存在的想法依赖性细胞因子，我们设计了以下特定目标：特定目的1：测试外泌体是否从耐受的四叠素基因敲除小鼠（CD81，CD63，CD9）的血清中分离出来的EBI3和P35 阳性外泌体。特定目的2：检查来自耐受CD81，CD63还是CD9的淋巴细胞是敲除小鼠产生并释放功能性IL35+免疫抑制外泌体。成功该提案中概述的具体目标的完成将填补我们对细胞因子IL35，理想情况下将导致未来的应用，进一步了解我们对治疗的理解 IL35对人类的影响。